Proteins with repetitive bacterial-ig-like (big) domains present in leptospira species

ABSTRACT

The invention relates to three isolated DNA molecules that encode for proteins, BigL1, BigL2 and BigL3, in the  Leptospira  sp bacterium which have repetitive Bacterial-Ig-like (Big) domains and their use in diagnostic, therapeutic and vaccine applications. According to the present invention, the isolated molecules encoding for BigL1, BigL2 and BigL3 proteins are used for the diagnosis and prevention of infection with  Leptospira  species that are capable of producing disease in humans and other mammals, including those of veterinary importance.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. application Ser. No. 13/525,157,filed Jun. 15, 2012, which is a divisional of U.S. application Ser. No.13/359,354, filed Jan. 26, 2012, now U.S. Pat. No. 8,216,594, issuedJul. 10, 2012, which is a divisional of U.S. application Ser. No.13/216,214, filed Aug. 23, 2011, now U.S. Pat. No. 8,124,110, issuedFeb. 28, 2012, which is a divisional of U.S. application Ser. No.13/078,879, filed Apr. 1, 2011, now U.S. Pat. No. 8,021,673, issued Sep.20, 2011, which is a divisional of U.S. application Ser. No. 12/728,177,filed Mar. 19, 2010, now U.S. Pat. No. 7,935,357, issued May 3, 2011,which is a divisional of application Ser. No. 11/332,464, filed Jan. 17,2006, now U.S. Pat. No. 7,718,183, issued May 18, 2010, which is adivisional of U.S. application Ser. No. 11/005,565, filed Dec. 7, 2004(abandoned); which is a divisional of U.S. application Ser. No.10/147,299, filed May 17, 2002 (abandoned). The entire content of eachof the earlier applications is hereby incorporated by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.AI001605, AI034431, HL051967, and TW000905 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “Sequence_Listing.txt”, created on Apr. 19, 2013, and having asize of 72.5 kilobytes, as permitted under 37 CFR 1.821(c). The materialin the aforementioned file is hereby incorporated by reference in itsentirety.

FIELD

The invention relates to three isolated DNA molecules that encode forproteins, BigL1, BigL2 and BigL3, in the Leptospira sp bacterium whichhave repetitive Bacterial-Ig-like (Big) domains and their use indiagnostic, therapeutic and vaccine applications. According to thepresent invention, the isolated molecules encoding for BigL1, BigL2 andBigL3 proteins are used for the diagnosis and prevention of infectionwith Leptospira species that are capable of producing disease in humansand other mammals, including those of veterinary importance.

BACKGROUND

Spirochetes are motile, helically shaped bacteria and include threegenera, Leptospira, Borrelia and Treponema, which are pathogens ofhumans and other animals. Borrelia and Treponema are the causativeagents of diseases that include Lyme disease, relapsing fever, syphilisand yaws. Leptospira consists of a genetically diverse group of eightpathogenic and four non-pathogenic, saprophytic species (1, 2).Leptospires are also classified according to serovar status—more than200 pathogenic serovars have been identified. Structural heterogeneityin lipopolysaccharide moieties appears to be the basis for the largedegree of antigenic variation observed among serovars (1, 2).

Leptospirosis is a zoonotic disease: transmission to humans occursthrough contact with domestic or wild animal reservoirs or anenvironment contaminated by their urine. Infection produces a widespectrum of clinical manifestations. The early-phase of illness ischaracterized by fever, chills, headache and severe myalgias. Diseaseprogresses in 5 to 15% of the clinical infections to produce severemultisystem complications such as jaundice, renal insufficiency andhemorrhagic manifestations (1-4). Severe leptospirosis is associatedwith mortality rates of 5-40%.

Leptospirosis has a world-wide distribution. Because of the largespectrum of animal species that serve as reservoirs, it is considered tobe the most widespread zoonotic disease (1). Leptospirosis istraditionally an important occupational disease among risk groups suchas military personnel, farmers, miners, sewage and refuse removalworkers, veterinarians and abattoir workers (1-3). However, new patternsof disease transmission have emerged recently that emphasize the growingimportance of leptospirosis as a public health problem. In developedcountries, leptospirosis has become the cause of outbreaks associatedwith recreational activities (1) and sporting events (1, 4, 5). InBrazil and other developing countries, underlying conditions of povertyhave produced large urban epidemics of leptospirosis associated withhigh mortality (4, 5).

In addition to its public health impact, leptospirosis is a majoreconomic burden as the cause of disease in livestock and domesticanimals (2). Leptospirosis produces abortions, stillbirths, infertility,failure to thrive, reduced milk production and death in animals such ascows, pigs, sheep, goats, horses and dogs and induces chronic infectionand shedding of pathogenic leptospires in livestock (2) and thereforerepresents an additional source of economic loss for the animalhusbandry industry because of current international and nationalquarantine regulations.

The control of human and animal leptospirosis is hindered by the currentlack of adequate diagnostic tools. The standard serologic test, themicroscopic agglutination test (MAT), is inadequate for rapid caseidentification since it can only be performed in few referencelaboratories and requires analyses of paired sera to achieve sufficientsensitivity (1, 2). Dependence upon the MAT results in delays inestablishing the cause of outbreaks as seen in several investigations(1, 2). Enzyme-linked immunosorbent assays (ELISA), and other rapidserologic tests based on whole-cell leptospiral antigen preparationshave been developed for use as an alternative method to screen forleptospiral infection, although the MAT is still required for caseconfirmation (1, 2). Recombinant antigen-based serologic tests arewidely used in screening for spirochetal infections such as Lyme diseaseand syphilis, but the use of recombinant proteins for serodiagnosis ofleptospirosis has not been widely investigated. Recently, a recombinantflagellar-antigen immuno-capture assay was described for serodiagnosisof bovine leptospirosis (6). A recombinant heat shock protein, Hsp58,showed a high degree of ELISA reactivity with serum samples from a smallnumber of human cases (7). However, the utility of recombinant antigensfor the serodiagnosis of leptospirosis has not been investigated inlarge validation studies.

Furthermore, there are no effective interventions presently available,which control or prevent leptospirosis. Environmental control measuresare difficult to implement because of the long-term survival ofpathogenic leptospires in soil and water and the abundance of wild anddomestic animal reservoirs (1, 3). Efforts have focused on developingprotective immunization as an intervention against leptospirosis.Currently-available vaccines are based on inactivated whole cell ormembrane preparations of pathogenic leptospires and appear to induceprotective responses through induction of antibodies against leptospirallipopolysaccharide (1, 3). However, these vaccines do not inducelong-term protection against infection. Furthermore, they do not providecross-protective immunity against leptospiral serovars that are notincluded in the vaccine preparation. The large number of pathogenicserovars (>200) and the cost of producing a multi-serovar vaccine havebeen major limitations in developing efficacious vaccines throughstrategies based on whole cell or membrane preparations.

The mechanism of pathogenesis in leptospirosis, as in spirochetaldisease such as Lyme disease and syphilis, relies on the pathogen'sability to widely disseminate within the host during the early stage ofinfection (2). Membrane-associated leptospiral proteins are presumed tomediate interactions that enable entry and dissemination through hosttissues. Putative surface-associated virulence factors serve ascandidates for vaccine strategies that induce responses to these factorswhich block dissemination in the host. Furthermore, membrane-associatedproteins would be accessible to the immune response during hostinfection and therefore, constitute targets for immune protectionthrough mechanisms such as antibody-dependent phagocytosis andcomplement-mediated killing. Production of these antigen targets asrecombinant proteins offers a cost-effective approach for protectiveimmunization for leptospirosis as a sub-unit based vaccine. In addition,selection of surface-associated targets that are conserved amongpathogenic leptospires can avoid the limitations encountered withcurrently available whole-cell vaccine preparations.

A major limitation in the field of leptospirosis has been identifyingsurface-associated and host-expressed proteins with conventionalbiochemical and molecular methods. From the genome sequence of thespirochete, Borrelia burgdorferi, more than 100 surface associatedlipoproteins were identified. Based on genome size and the biology ofits lifecycle, Leptospira are expected to have a significantly greaternumber of surface-associated targets. At present, less than 10surface-associated proteins have been characterized through isolation ofmembrane extracts, purification and characterization of proteins inthese extracts and molecular cloning of these protein targets (8-14)(12). Immunization with recombinant proteins for several identifiedtargets, LipL32, OmpL1 and LipL41, induce partial, but not complete,protective responses (11, 12).

To develop a more comprehensive understanding of leptospiral proteinexpression we have used the humoral immune response during humanleptospirosis as a reporter of protein antigens expressed duringinfection. The identification of leptospiral antigens expressed duringinfection has potentially important implications for the development ofnew serodiagnostic and immunoprotective strategies. Sera from patientswith leptospirosis was used to identify clones from a genomic LeptospiraDNA phage library which express immunoreactive polypeptides. Aproportion of these clones were found to encode a novel family ofmembrane-associated Leptospira proteins. The identification of thesepolynucleotides and polypeptides and their application for diagnosis ofleptospirosis and inducing an immune response to pathogenic spirochetesis the basis for this invention.

SUMMARY

The invention relates to DNA molecules in Leptospira and thepolypeptides they encode which have repetitive bacterial Ig-likedomains. The invention describes the isolation of three DNA molecules,originally derived from L. kirschneri and L. interrogans, which encodeproteins, herein designated “BigL1”, “BigL2” and “BigL3”, that havemolecular masses of approximately 110, 205 and 205 kDa, respectively,based on the predicted amino acid sequence of the polypeptides. Thethree proteins have 12-13 tandem repeat sequences of approximately 90amino acids. Repeat sequences from BigL1, BigL2 and BigL3 are highlyrelated (>90% amino acid sequence identity) to each other and belong tothe family of bacteria Ig-like (Big) domains, moieties which are foundin virulence factors of bacterial pathogens.

The DNA molecules that encode for Leptospira proteins with Big domains,herein called “bigL1”, “bigL2” and “bigL3”, can be inserted asheterologous DNA into an expression vector for producing peptides andpolypeptides. Recombinant polypeptides can be purified from surrogatehosts transformed with such expression vectors. BigL1, BigL2 andBigL3-derived polypeptides are serological markers for active and pastinfection since sera from leptospirosis patients and animals infected orimmunized with pathogenic Leptospira recognize isolated polypeptides.

Furthermore, BigL1, BigL2 and BigL3 polypeptides from recombinant ornative antigen preparations are immunogenic. Antibodies obtained fromexperimental animals immunized with purified recombinant BigL1, BigL2and BigL3 polypeptides recognize native antigen from Leptospira, and areuseful for detecting pathogenic spirochetes in samples from subjectswith a suspected infection.

In addition, BigL1, BigL2 and BigL3 polypeptides induce an immuneresponse against pathogenic spirochetes. BigL1, BigL2 and BigL3-derivedpolypeptides; antibodies to these polypeptides; and polynucleotides thatencode for BigL1, BigL2 and BigL3 may be used alone or combined withpharmaceutically acceptable carrier to treat or prevent infection withLeptospira. Since Big domains are present in proteins associated withvirulence in other bacterial pathogens, these moieties may be used totreat or prevent infections unrelated to those caused by Leptospira.

In a first embodiment, the invention provides isolated DNA molecules forbigL1, bigL2 and bigL3 and the polypeptides that are encoded by theseDNA molecules or have functionally equivalent sequences. In addition, amethod is provided for producing an expression vector containing bigL1,bigL2 and bigL3 polynucleotides and obtaining substantially purifiedpolypeptides derived from these sequences.

A second embodiment of the present invention is to providepharmaceutical composition for inducing immune responses in subjects topathogenic spirochetes, comprising an immunogenically effective amountof one or more selected antigens among the group consisting of BigL1,BigL2, BigL3 and polypeptides with functionally equivalent sequences ina pharmaceutically acceptable vehicle.

In a third embodiment, the invention provides a method for identifying acompound which binds to BigL1, BigL2, BigL3 polypeptides or polypeptideswith functionally equivalent sequences that includes incubatingcomponents comprising the compound and BigL1, BigL2 or BigL3 polypeptideor polypeptides with functionally equivalent sequences under conditionssufficient to allow the components to interact and measuring the bindingof the compound to the BigL1, BigL2 or BigL3 polypeptide or polypeptideswith functionally equivalent sequences. Preferably, the inventive methodis a serodiagnostic method utilizing sera from a subject with asuspected active or past infection with Leptospira or other relatedbacterial pathogen.

In a fourth embodiment, the invention provides a method for detectingpathogens in a sample which includes contacting a sample suspected ofcontaining a pathogenic spirochete with a reagent that binds to thepathogen-specific cell component and detecting binding of the reagent tothe component. In one aspect, the reagent that binds to thepathogen-specific cell component is an oligonucleotide for theidentification of bigL1, bigL2 and bigL3 polynucleotide. In anotheraspect, the reagent that binds to the pathogen-specific cell componentis an antibody against the BigL1, BigL2 or BigL3 polypeptide orpolypeptides with functionally equivalent sequences.

In a fifth embodiment, the invention provides a kit useful for thedetection of BigL1, BigL2, and BigL3 polypeptides or polypeptides withfunctionally equivalent sequences; bigL1, bigL2 and bigL3polynucleotides; or antibodies that bind to BigL1, BigL2, BigL3,polypeptides or polypeptides with functionally equivalent sequences.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B show a Southern blot analysis of bigL gene sequences inLeptospira. Genomic DNA (3 mcg/lane) from L. interrogans strain FiocruzL1-130 (lanes 1), L. kirschneri strain Rm52 (lanes 2) and L. biflexistrain Patoc I (lanes 3) digested with NsiI and subject to agarose gelelectrophoresis. After transfer to nitrocellulose membranes, blots wereprobed with DNA fragments that encode for BigL repetitive domains(4^(th)-6^(th) repetitive domain of BigL3, FIG. 1A) and C-terminalregions of bigL1, bigL2 and bigL3, which are unique to each of thesegenes, respectively (FIG. 1B).

FIG. 2 shows a schematic diagram of the genomic organization of theregion encoding the BigL1 and BigL3 proteins in L. kirschneri. The BigL1protein would contain a signal peptide (hatched box) and thirteen90-amino-acid bacterial immunoglobulin-like domains (solid boxes). TheBigL3 protein would contain a signal peptide, twelve 90-amino-acidbacterial immunoglobulin-like domains, and a 793 amino acidcarboxyterminal (C-terminal) domain. The locations of the 2156 bp regionof 100% DNA sequence identity are shown. The organization of the regiondepicted was conserved in L. interrogans and L. kirschneri.

FIG. 3 shows the polymerase chain reaction (PCR) amplification of DNAfragments from strains of five pathogenic species of Leptospira.Degenerate primers were designed based on the L. kirschneri and L.interrogans sequence encoding for the BigL3 region corresponding topositions 46-65 aa. PCR reactions were performed from purified DNA fromfive pathogenic (L. kirschneri, borgpetersenii, interrogans, santarosai,and noguchi) and two non-pathogenic species (L. biflexi and wolbachii).

FIG. 4 shows amplified products from RT-PCR of RNA extracts of L.kirschneri with bigL1, bigL2 and bigL3 specific primers. Reversetranscription reactions (lanes “+”) were performed on RNA extracts ofcultured leptospires and then subject to a polymerase chain reaction(PCR) amplification step with primers that bind to unique sequenceswithin bigL1, bigL2 and bigL3. Amplification with primers based onsequences within lipL45 was performed as a control reaction as were PCRreactions for which samples were not subjected to the reversetranscription step.

FIG. 5 shows the immunoblot reactivity of pooled sera from patients andanimal reservoirs infected with pathogenic Leptospira and laboratoryanimals immunized with whole L. interrogans antigen preparation torecombinant BigL3 protein (rBigL3). Western blot analysis was performedwith purified rBigL3 (1 mcg per lane, lanes 3). Membranes were probedwith sera from patients with leptospirosis (lane A), healthy individuals(lane B), captured rats that are colonized with L. interrogans (lane C),captured rats that are not colonized with L. interrogans (lane D),laboratory rats immunized with whole antigen preparations of in vitrocultured L. interrogans (lane E) and pre-immune sera from the laboratoryrats collected prior to immunization (lane F). Reactivity to whole L.interrogans antigen preparation (lanes 1) and recombinant LipL32 protein(rLipL32, lanes 2) is shown for comparison. The numbers on the leftindicate the positions and relative mobilities (kDa) for molecular massstandards (Invitrogen).

FIG. 6 shows an ELISA evaluation of individual patient seroreactivity torBigL3 during the acute (lanes A) and convalescent (lanes B) phase ofillness with leptospirosis. Sera from 4 leptospirosis patients (unbrokenlines) and 4 healthy individuals (broken lines), at dilutions of 1:50,1:100 and 1:200, were incubated with RBigL3 (25-200 ng/well). Mu andgamma chain specific antibodies conjugated to horseradish peroxidasewere used to determine IgM and IgG seroreactivity, respectively. Meanabsorbance values (OD 450 nm) and standard deviations are represented inthe graphs.

FIG. 7 shows the rBigL3 IgM (Column A) and IgG (Column B) reactivity ofsera from 29 individual patients with leptospirosis during the acute(lanes 2) and convalescent (lanes 3) phase of illness and 28 healthyindividuals (lanes 1). Sera (1:50 dilutions) and Mu and gamma chainspecific antibodies conjugated to horseradish peroxidase were used todetermine reactivity. Solid bars represent mean absorbance (OD 450 nm)values.

FIG. 8 shows the immunoblot reactivity of individual patients withleptospirosis to rBigL3 during the acute (lanes 6-9) and convalescent(lanes 10-13) phase of illness. Western blot analysis was performed withpurified rBigL3 (1 mcg per lane, lanes 3). Membranes were probed withsera diluted 1:100. Gamma chain-specific antibodies conjugated toalkaline phosphatase were used to determine reactivity to therecombinant 58 kD protein of region 1 of BigL3 (2^(nd) to 6^(th) Bigrepeat domains). Reactivity to rLipL32 (1 mcg per lane) was performed asa comparison. The mobility of purified rBigL32 and rLipL32 (lane 14) andmolecular mass standards (lane 15) are shown after staining withPonceau-S and Coomassie blue, respectively.

FIG. 9 shows the immunoblot reactivity of rat anti-rBigL3 antisera torBigL3 and native antigen from L. interrogans lysates. Immunoblots wereprepared with purified rBigL3 (1 mg/lane; lanes 3, 5, 7, 9) and wholeantigen preparations (10⁸ leptospira per lane; lanes 2, 4, 6 and 8) fromcultured leptospires. Membranes were probed with pooled sera (dilutions1:500 [lanes 4 and 5], 1:100 [lanes 6 and 7] and 1:2500 [lanes 8 and 9])from rats immunized with rBigL3 from E. coli expressing a cloned DNAfragment of bigL3 from L. interrogans. Pre-immune sera was obtainedprior to the first immunization and used in the immunoblot analysis as acontrol (lanes 2 and 3). The mobility (kDa) of molecular mass standardsare shown on the left side of the figure.

FIG. 10 shows the immunoblot reactivity of rabbit anti-rBigL3 antiserato native antigen from Leptospira strain lysates. Immunoblots wereprepared with whole antigen preparations (10⁸ leptospira per lane) ofthe following cultured strains: lane 1, L. interrogans sv pomona (typekennewicki) strain RM211, low-passage; lane 2, L. interrogans svcanicola strain CDC Nic 1808, low passage; lane 3, L. interrogans svpomona strain PO-01, high passage; lane 4, L. interrogans sv bratislavastrain AS-05, high passage; lane 5, L. kirschneri sv grippotyphosastrain RM52, low passage; lane 6, L. kirschneri sv grippotyphosa strainP8827-2, low passage; lane 7, L. kirschneri sv grippotyphosa strain86-89, low passage; lane 8, L. kirschneri sv grippotyphosa strain MoskvaV, high passage; lane 9, L. kirschneri sv mozdok strain 5621, highpassage; lane 10, L. kirschneri sv grippotyphosa strain RM52, highpassage. Membranes were probed with sera from rabbits immunized withrBigL3 from E. coli expressing a cloned DNA fragment of bigL3 from L.kirschneri and, as a control measure, sera from rabbits immunized withrecombinant L. kirschneri GroEL protein. The positions of nativeantigens corresponding to BigL3 and GroEL and the mobility (kDa) ofmolecular mass standards are shown on the left and right sides,respectively, of the figure.

DETAILED DESCRIPTION

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BigL—are polypeptides of Leptospira sp. having tandem repeat sequenceseach of which are similar, according to their sequence homology, tobacterial immunoglobulin-like (Big) domains. Big domains are present inbacterial proteins, expressed in bacterial pathogens such as E. coli,Yersinia and Bordetella, which have virulence functions such as hostcell adhesion.

Reference sequence—is a new sequence obtained by isolation from anatural organism or through genetic engineering and presents an accuratebiological function, which is characteristic of the present invention.

Functionally equivalent sequences—are the sequences, related to areference sequence, that are the result of variability, i.e. allmodification, spontaneous or induced, in a sequence, being substitutionand/or deletion and/or insertion of nucleotides or amino acids, and/orextension and/or shortening of the sequence in one of their ends,without resulting in modification of the characteristic function of thereference sequence. Functionally equivalent sequences encompassfragments and analogs thereof. In other words, sequences functionallyequivalent are sequences that are “substantially the same” or“substantially identical” to the reference sequence, such aspolypeptides or nucleic acids that have at least 80% homology inrelation to the sequence of amino acids or reference nucleic acids. Thehomology usually is measured by a software system that performs sequenceanalyses (Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710, UniversityAvenue, Madison, W is, 53705).

As we mentioned before, Leptospira antigens expressed during the hostinfection are important in the identification of targets for diagnosistests and vaccines. The LipL32 protein is one of these targets and wasidentified as immunodominant antigen by the immune humoral responseduring the natural infection. However the sensitivity of serologic testsbased upon detection of antibodies against LipL32 in patient sera duringacute-phase illness with leptospirosis detection is limited (seeFlannery, B: “Evaluation of recombinant Leptospira antigen-basedEnzyme-linked Immunosorbent Assays for the serodiagnosis ofLeptospirosis” J. Clin. Microbiology 2001; 39(9): 3303-3310; WO9942478).

The present invention is based on the identification of the family ofproteins BigL associated with species of spirochetal bacteria, includingthose belonging to Leptospira.

According to the present invention, the BigL protein family wasidentified as targets of the host humoral immune response, generatedduring infection with pathogenic Leptospira or immunization withpathogenic Leptospira or recombinant BigL polypeptides. BigLpolypeptides and polynucleotides that encode these polypeptides areuseful as in diagnostic tests to identify naturally occurring infectionin different species including humans and animal reservoirs. Thediagnostic test based on those proteins presents improved sensitivityand specificity in relation to standard diagnostic tests or those thathave been used in the published literature. The identification ofleptospirosis in the initial phase. In addition BigL polypeptides caninduce immune responses when used in a pharmaceutical composition forimmunization.

In the present invention, the three BigL polypeptides are characterizedwith molecular weights 128.4 kD, 201.3 kD and 200.4 kD, based on thededuced amino acid sequence of the isolated polynucleotides, bigL1,bigL2 and bigL3, which encode for these polypeptides. The amino acidsequence of the BigL polypeptides has a signal sequence and a putativesignal peptidase cleavage site largely conforming to the spirochetallipobox; therefore BigL polypeptides are membrane-associatedlipoproteins. The polypeptides of 128.4 kD, 201.3 kD and 200.4 kD aredesignated “BigL1”, “BigL2” and “BigL3”, respectively.

Although the BigL polypeptides of the present invention have beenisolated originally of Leptospira sp, they are useful not just forinduction of the immune response against the pathogenic organismsLeptospira sp., but also against other spirochetes bacteria andpathogens that have factors with Big domains. Additionally, BigLpolypeptides can be used for the diagnosis of infections due toLeptospira sp., other pathogenic spirochetes and bacterial pathogens.

Several processes that incorporate state-of-the-art methodologies can beused to obtain polynucleotide sequences that encode for BigLpolypeptides. These processes include, but they are not limited to, theisolation of DNA using hybridization of genomic libraries with probes todetect homologous sequences of nucleotides; screening of antibodies ofexpression libraries to detect fragments of cloned DNA with sharedstructural aspects; polymerase chain reaction (PCR) in genomic DNA usinginitiators able to recombine sequence of DNA of interest; andcomputer-based searches of sequence databases for similar sequences tothat of the bigL polynucleotides.

In the present invention the identification of the antigens was based onknowledge that there is differential expression of Leptospira antigensduring culture (in vitro) and during host infection (in vivo).Differential expression of Leptospira antigens is presumed to beimportant in host adaptation during infection. We used a strategy toidentify immunoreactive antigens and therefore antigens expressed duringhost infection. Sera from patients infected with pathogenic Leptospirawere used to select polynucleotide sequences from genomic Leptospira DNAlibrary in lambda phage that encode for immunoreactive polypeptides.

The present invention identified and isolated three polynucleotides withnucleotide sequences corresponding to SEQ ID No:1, SEQ ID No:3 and SEQID No:5, as well as the amino acid sequences of the respectivepolypeptides, BigL1, BigL2 and BigL3, encoded by such nucleotides.

Step 1

The screening the positive clones consisted basically of the followingsteps:

(a) The DNA of a pathogenic Leptospira was cut with an appropriateenzyme and ligated into a specific site in the lambda phage genome. Hostbacteria were infected with phage and the resulting clones, expressingrecombinant polypeptides after induction with IPTG, were submitted toimmunoblot protocol where a membrane of colony lysates was incubatedwith sera from patients with laboratory confirmed leptospirosis and thenwith a secondary antibody conjugated to horseradish peroxidase, whichrecognized human Ig. Positive clones were detected through an indicatorreaction, for antigen-antibody complexes based on the production ofcolor.(b) The sequence of cloned and isolated polynucleotides was determinedusing phage vector-specific sequences as initiators of the sequencingreaction. Analysis of the clone sequences and the use of a primerwalking strategy identified the complete nucleotide sequence for thegenes that encode for BigL1, BigL2, and BigL3.(c) Most of the obtained positive clones contain genes encoding proteinsof thermal shock Hsp58 and DnaK and the protein of outer membraneLipL41. However, it was found clones containing genes encodingrepetitions in tandem of 90 amino acids compared by Database of proteinsfamily (Pfam) as proteins of bacterium, type immunoglobulin (Big). Withthe analysis of the clone sequences, were identified 3 genes containing12 tandem repeats for bigL1 and 13 tandem repeats in bigL2 and bigL3.

Step 2—Subcloning Expression and Purification of the Protein

Drawing of two oligonucleotides with base in sequences of two proteinsBigL

Amplification by PCR of the initial BigL portion encoding for part ofthe repetitive region, from those oligonucleotides

Sequencing of the product of the amplification

Subcloning of the region-encoding by the product sequenced

Expression of the recombinant protein.

Purification of the recombinant protein.

Immunoblot analyses demonstrate that sera from leptospirosis patient androdent reservoirs infected with pathogenic Leptospira produce antibodiesprimarily to the BigL domain repeats of the BigL polypeptides,indicating that they are the main antigenic regions recognized duringinfection.

In relation to the polypeptides of the present invention they consist ofsequences of DNA, cDNA or RNA (and sequences of nucleic acids which arecomplementary), as well as their functionally equivalent sequence, i.e.,those sequences that encode the whole or a part, of the polypeptidesdesignated as BigL1, BigL2 and BigL3, but are non-identical due tovariability.

The polypeptides and polynucleotides in the present invention consist ofBigL1, BigL2 and BigL3 and the polynucleotides that encode thesepolypeptides; however they include, in addition, polypeptides andpolynucleotides that have functionally equivalent sequence.

In the present invention, both polynucleotides and polypeptides may beof natural, synthetic or recombinant origin, having the necessary puritydegree to grant to their biological activities.

The present invention also refers to the polynucleotides encoding forBigL1, BigL2 and BigL3 which are used in PCR reactions to obtain eithercomplete or partial amplified DNA fragments of the bigL polynucleotides,for the purpose of detection of Leptospira in samples or expression ofrecombinant BigL polypeptides. In the case of initiators used for thepolynucleotide amplification in the present invention, they areoligonucleotides made of two or more deoxyribonucleotides orribonucleotides, natural or synthetic.

Each initiator is preferably constructed in order to be substantiallysimilar to a flanking region of the sequence strand that is the targetfor amplification. In this sense, an initiator can be designatedfunctionally equivalent if corresponding polymers can produce the sameprocess, without being identical, facing the utilization or applicationconsidered.

Polynucleotide sequences of this invention can also be inserted in anexpression vector, such as a plasmid, virus or other vehicle used forrecombinant cloning, which is used by inserting or incorporating wholeor partial nucleotide sequences that encode for BigL1, BigL2 and BigL3or their functionally equivalent sequences. Such expression vectorscontain a promoter sequence that facilitates the efficient transcriptionfrom genetic sequence in the host in which the vector is inserted. Suchhosts can include prokaryotes or eukaryotes, including microorganismssuch as yeast or insects and mammals. Such processes for the use ofexpression vectors construction and for the expression of recombinantsequences, properly so-called, are well known by experts in technique.

The present invention provides for a method to produce antibodies thatbind to complete or partial polypeptides of BigL1, BigL2 and BigL3 ortheir functionally equivalent sequences. Such antibodies are useful asresearch and diagnostic tools in the study and diagnosis of spirocheteinfections in general, and more specifically in the development ofdiagnostics and therapeutics whether treatment or prevention, forleptospirosis. Such antibodies can be administered alone or as part of apharmaceutical composition that uses these antibodies and apharmaceutically acceptable carrier as part of anti-spirochetaltherapeutic.

The invention relates to the use of pharmaceutical compositions of BigLpolypeptides or the polynucleotides that encode for these polypeptidesas vaccines, either as a vaccine for prevention of disease which inducesan immunoprotective response to infection or colonization withpathogenic spirochetes or as therapeutic vaccine that provides abeneficial impact in reducing the duration or severity of the clinicalcourse of illness in a subject due infected with a pathogenic spirocheteor in reducing the reservoir state of a carrier of pathogenic spirochetesuch as in pigs, cows, rats or dogs that harbor and excrete pathogenicspirochetes for prolonged periods of time. Such compositions may beprepared with an immunogenically effective quantity of an antibodyagainst BigL1, BigL2 and BigL3 respectively, or with one or more ofBigL1, BigL2 and BigL3 isolated from the leptospiral pathogen orrecombinant BigL polypeptides, or their functionally equivalentsequences, in excipients and additives or auxiliaries.

Another embodiment of present invention relates to the pharmaceuticalcomposition used to induce an immune response to a pathogenic spirochetein an individual, particularly Leptospira sp., including aimmunologically effective quantity of BigL1, BigL2 and BigL3 or of theirfunctionally equivalent sequence in a pharmaceutically acceptablevehicle. As “individual” we refer to any mammal, including humans,rodents, domesticated and laboratory animals and livestock. As “quantityimmunologically effective” we refer to quantity of BigL polypeptideantigen necessary to induce, in an individual, an immunological responseagainst Leptospira or any other pathogenic spirochete or bacterialpathogen. The invention further provides a kit for:

1—detecting one of polypeptides, BigL1, BigL2 and BigL3, or theirfunctionally equivalent sequences;2—detecting nucleic acid encoding for BigL1, BigL2 and BigL3 or theirfunctionally equivalent sequences;3—detecting antibodies for such polypeptides, BigL1, BigL2 and BigL3, ortheir functionally equivalent sequences.

The kit used for detection of BigL polypeptides includes those that usea vehicle containing one or more receptacles with a first receptaclecontaining a linking reagent to BigL1, BigL2 and BigL3 or to theirfunctionally equivalent sequences.

The kit used for detection of polynucleotides that encode BigLpolypeptides includes those that use a vehicle containing one or morereceptacles with a first receptacle containing a polynucleotide thathybridizes to the nucleic acid sequence that encodes BigL1, BigL2 andBigL3 or to their functionally equivalent sequences.

The kit useful for detecting antibodies against BigL polypeptidesincludes those that use a vehicle containing one or more receptacleswith a first receptacle containing a polypeptide of BigL1, BigL2 andBigL3 or of their functionally equivalent sequences.

The present invention will be now described with reference to theExamples, which should not be considered as limiting of the presentinvention.

Example 1 Example 1A Bacterial Strains, Plasmids and Media

Leptospira kirschneri serovar grippotyphosa, strain RM52, was isolatedduring an outbreak of porcine abortion in 1983. L. interrogans serovarcopenhageni, strain Fiocruz (L1-130), was isolated from the bloodstreamof a human leptospirosis patient. L. kirschneri serovar grippotyphosastrain RM52 and other leptospiral strains were obtained from theNational Leptospirosis Reference Center (National Animal Disease Center,Agricultural Research Service, U.S. Department of Agriculture, Ames,Iowa). Leptospiral strains were cultivated at 30° C. in Johnson-Harrisbovine serum albumin-Tween80 medium (Bovuminar PLM-5 MicrobiologicalMedia, Intergen (2)). Low-passage samples of the RM52 isolate wereeither stored in liquid nitrogen or passaged in liquid medium at least200 times to generate a high-passage form. The high-passage strain wasunable to produce a lethal infection in hamsters at any dose and wasonly able to infect hamsters at a dose of 10⁷ by intraperitonealinoculation.

Escherichia coli XL1-Blue MRF′ ΔmcrA)183ΔmcrCB-hsdSMR-mrr)173 endA1supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn10 (Tetr)](Stratagene) and E. coli PLK-F′ (endA1 gyrA96 hsdR17 lac⁻ recA1 relA1supE44 thi-1 [F′ lacI^(q)ZΔ15]) were used as the host strains forinfection with the λZap II (Stratagene) and λTriplEx (Clontech) vectors,respectively. E. coli SOLR (e14⁻[mcrA], Δ[mcrCB-hsdSMR-mrr]171 sbcC recBrecJ umuC::Tn5[Kan^(r)] uvrC lac gyrA96 relA1 thi-1 endA1 λ^(r), [F′proAB lacI^(q)ZΔM15], Su⁻ [non-suppressing]] and E. coli BM25.8 (supE44thi Δlac-proAB [F′ traD36 proAB⁺ lacI^(q)ZΔM15] λimm434(kan^(r))P1(cam^(r)) hsdR(r^(K12−)m^(K12−))) were used for in vivo excision ofthe pBluescript and pTriplEx phagemids, respectively. BLR(DE3)pLysS [F⁻ompT hsdS_(B) (r_(B)-m_(B)-) gal dcm _(srl-recA)306::Tn10(TcR) (DE3)pLysS(CmR)] (Novagen) was used as the host strain for the pRSETexpression vector (Invitrogen). E. coli strains were grown in LBsupplemented with 100 μg/ml ampicillin, 100 μg/ml carbenicillin, or 25μg/ml chloramphenicol where appropriate. Antibiotics were purchased fromSigma.

Example 1B Isolation and Characterization of bigL Genes

This example illustrates the identification and isolation of the bigLgenes. Genomic DNA was prepared from virulent, low-passage L.kirschneri, serovar grippotyphosa, strain RM52 by the method of Yeltonand Charon (15). Genomic DNA was prepared from a clinical isolate of L.interrogans, serovar copenhageni, strain Fiocruz L1-130, using a kit togenomic DNA (Qiagen). The QIAquick PCR Purification Kit (Qiagen) wasused to obtain purified DNA. The genomic DNA was partially digested withTsp509I and ligated to λTriplEx arms following the instructions provided(Clontech). The Gigapack III Gold Packaging Extract (Stratagene) wasused to package ligated, digested genomic DNA into lambda heads. Thephage titer of the library was determined by infection E. coli XL1-Blue.

For screening of the genomic library, approximately 10³ pfu were platedon a lawn of E. coli XL1-Blue, transferred to nitrocellulose membrane(Schleicher & Schuell), sensibilized with IPTG and processed asrecommended (Schleicher & Schuell). The nitrocellulose filter wasblocked with 5% skimmed milk in Tris-buffered saline (pH7.8) with 0.05%Tween 20 (TBST) or phosphate-buffered saline (pH 7.4) with 0.05% Tween20 (PBST), and incubated for 1 hour with pooled sera, diluted 1:50, frompatients with laboratory-confirmed leptospirosis. Sera were collectedfrom patients, identified in urban epidemics in Brazil between 1996 and1999, during the convalescent-phase of illness, and were pre-absorbedwith E. coli lysates prior to use to remove antibodies to E. coli.Membranes were washed three times with TBST or PBST, and incubated formore than 1 hour with rabbit or goat anti-human immunoglobulin antibodyconjugated with alkaline phosphatase (Sigma) in the dilution of 1:1000.Detection with NBT (0.3 mg/ml) and BCIP (0.15 mg/ml) or development withthe ECL Western Blot Detection Reagents (Amersham) followed by exposureto Hyperfilm (Amersham) was used to identify plaques withantigen-antibody complexes.

Each positive plaque was stored at 4° C. in 1 ml SM (0.1 M NaCl, 8 mMMgSO₄, 50 mM Tris-HCl pH 7.5; 0.01% in gelatin, with 1-2 drops ofchloroform. The lambda plaque clones that reacted with pooled sera weresubjected to two additional stages of purification. The genomic DNAfragments inserted into lambda bacteriophage were excised by infectingE. coli SOLR or BM25.8 strains with the lambda clones as described bythe supplier (Stratagene and Clontech, respectively).

The sequence of the first 500-700 nucleotides of the insert was obtainedusing a vector-specific primer that links adjacent to the insert.Nucleotide sequence analysis of 131 clones identified 13 that had DNAfragment inserts, found to encode tandem repeats approximately 90 aminoacids in length. Each of the repeat sequences were subsequentlyidentified in Pfam 6.6 (available online at pfam.wustl.edu/) to belongto the Big2 family Big2 family of bacterial immunoglobulin-like (Big)domains.

To identify sequences that encode full-length proteins according to thepredicted amino acid sequence, the nucleotide sequences of the cloneswere assembled from individual sequences obtained by a combination ofprimer walking and sequencing of nested deletions. The deletions weregenerated from the plasmid clones by removal of restriction fragmentsextending from inside the insert into the multicloning sites flankingthe insert. Oligonucleotides were synthesized and obtained from GIBCOBRL or Operon. Inverse PCR (iPCR) was performed to obtain sequencescontaining the remainder of the genes and flanking DNA. The UCLA CoreSequencing Facility, the Yale/Keck Core DNA Sequencing Facility and theUniversity of California at Berkeley Sequencing Facility performed thesequencing reactions.

Two L. kirschneri clones and four L. interrogans clones were found toencode a gene which we designate bacterial immunoglobulin-likeLeptospiral protein one, bigL1. The complete nucleotide sequence of L.kirschneri bigL1 and the predicted amino acid sequence of the geneproduct is shown in SEQ ID NO: 1 and SEQ ID NO: 2. Six L. kirschnericlones were found to encode a second gene which we designated bigL2. Thecomplete nucleotide sequence of L. kirschneri bigL2 is shown in SEQ IDNO: 3. L. kirschneri bigL2 appears to be a pseudogene, an extra adenineresidue occurs at nucleotide 1011 resulting in a frameshift mutation anddownstream TAG stop codon. However, the antibody screening with pooledpatient sera was able to identify lambda clones with DNA fragmentsencoding bigL2 gene products, presumably since the cloned fragments didnot have the frameshift mutation and were inserted in an orientationthat allowed expression of a product that was recognized by patientsera. The predicted amino acid sequence of the L. kirschneri bigL2 geneproduct, without the frameshift mutation, is shown in SEQ ID NO: 4. Afifth L. interrogans clone was found to encode several Big repeatsinitially thought to belong to BigL1. However the upstream DNA encodedby this fifth L. interrogans clone was found to differ from the sequenceupstream of bigL1. Sequencing the regions flanking the bigL1 generevealed that the fifth L. interrogans clone corresponded to a thirdgene, designated bigL3, downstream of bigL1 (FIG. 2). The completenucleotide sequence for bigL3 was obtained from L. kirschneri DNA and isshown in SEQ ID NO: 5. The predicted amino acid sequence of the L.kirschneri bigL3 gene product is shown in SEQ ID NO: 6.

All three bigL genes encode a signal peptide and putative signalpeptidase cleavage site largely conforming to the spirochetal lipobox,as previously defined (Haake, D. A. 2000. Spirochetal lipoproteins andpathogenesis. Microbiology. 146:1491-1504). Comparison of the sequencesof known spirochetal lipoproteins indicates that the spirochetal lipoboxis much more loosely defined than the E. coli lipobox. For example,while most E. coli lipoproteins have Leu in the −3 position relative toCys, spirochetal lipoproteins may also have a number of otherhydrophobic amino acids in this position, including Val, Phe, and Ile.E. coli experiments involving site-specific mutagenesis of amino acidsfollowing cysteine indicate that acidic residues cause sorting oflipoproteins to the cytoplasmic membrane. Sequence analysis ofleptospiral lipoproteins indicates that a similar sorting signal ispresent in these bacteria. For example, LipL31 is the only lipoproteinhaving an unopposed negative charge in the first two amino acidsfollowing cysteine, and is also the only lipoprotein sorted exclusivelyto the cytoplasmic membrane. Like the outer membrane lipoproteins LipL32and LipL41, the BigL proteins have uncharged amino acids in the +2 and+3 positions, indicating that they would be sorted to the outermembrane.

Following their signal peptides, all three proteins would contain aseries of tandem repeats, approximately 90-amino-acids in length. Themature BigL1 protein would consist almost entirely of thirteen repeats,while in contrast BigL2 and BigL3 contain twelve repeats followed bylarge carboxy-terminal domains. Though there is a high degree ofsequence variation among the 31 unique repeats found in the threeproteins, all of the repeats were identified by the Pfam database asbacterial immunoglobulin-like Big protein family with E-values as low as4×e⁻³⁰.

The L. interrogans and L. kirschneri versions of bigL1, bigL2, and bigL3were highly related, with >90% DNA and amino acid sequence identity. Inboth species there is a region of DNA sequence identity involving the 5′ends of bigL1 and bigL3 (FIG. 2). The region of sequence identity beginsextends from the initial ATG start codon to position 1890 bp in bothgenes. The large region of DNA sequence identity between bigL1 and bigL3results in an identical amino acid sequence for the first 630 aminoacids (positions 1-630) of BigL1 (SEQ ID NO: 2) and BigL3 (SEQ ID NO:6). This region of identity corresponds to the first six BigL domainrepeats.

Example 2 Example 2A Characterization of the bigL Genes and Detection ofbigL DNA and RNA

This example illustrates the distribution of multiple copies of bigLgenes among Leptospira species and methods to detect bigL DNA and RNA insamples.

Southern Blot Analysis

Southern blot analysis was performed to identify multiple copies of bigLgenes in genomic DNA from L. interrogans strain Fiocruz L1-130, L.kirschneri strain RM52, and L. biflexi strain Patoc I. DNA restrictionand modifying enzymes were purchased from New England Biolabs. GenomicDNA was extracted from 500 ml of 7-day cultures of Leptospira cells withthe Blood and Cell Culture kit (Qiagen, Valencia, Calif.). Approximately3 mcg of DNA was digested with 5-20 units of NsiI overnight in a finalvolume of 50 mcl. DNA was then purified with phenol:chloroform:isoamyland precipitated with 100% cold ethanol and 3M sodium acetate pH andwashed with 70% ethanol. Purified DNA was then re-digested with 5-20units Pad overnight in a final volume of 25 mcl. The double digested DNAwas separated in a 0.8% agarose gel at 20V overnight. The gel was thenincubated twice for 30 minutes in denaturing buffer (1.5 M NaCl, 0.5 NNaOH), and twice for 30 minutes in neutralization buffer (1M Tris(pH7.4) 1.5 M NaCl). Genomic DNA was transferred onto a positivelycharged nylon membrane (Roche Molecular Biochemicals, Indianapolis,Ind.) according to the method described by Southern.

Probes were synthesized with the PCR Dig Probe Synthesis kit (Roche,Manheim, Germany). Reactions were assembled according to themanufacturer in a final volume of 50 mcl. Temperature cycles for theamplification were 94° C. for 5 min, 94° C. for 30 sec, 57° C. for 30 smin, and 72° C. for 1 min, with a final extension time of 7 min after atotal of 35 cycles. Probe sequences were as follows: to amplify the bigLDNA fragments that code for BigL repetitive domains, a bigL3 DNAsequence was selected that corresponds to the region that encodes forBigL3 repetitive domains 4-6, BigL3_(—)395 gat-ttt-aaa-gtt-aca-caa-gcand BigL3_(—)573 aaa-ccg-gac-tac-tta-cct-ttc-c; and to amplify bigL DNAfragments that are specific for each of the bigL genes, sequences thatencode for C-terminal regions of the BigL gene products were selected:BigL1.2078p, tta-cgg-cta-cag-gta-ttt-tta-cg and BigL1.2691patt-gga-aga-ttt-cca-agt-aac-c, BigL2.5121p tat-cta-cgc-tgc-aaa-tgg andBigL2.5865p ttg-ttg-gcg-ata-cgt-ccg, BigL3.5071pcat-aac-tct-cct-cat-aac-a and BigL3.5548p tat-gta-gag-ata-aga-tcc.

The UV Crosslinked membrane was subject to prehybridization at 42° C.for 1 hour in Dig Easy Hybridization solution (Roche). Prior tohybridization, the Dig labeled probes were boiled for 10 minutes andrapidly transferred to ice for 5 minutes. The denatured probes weremixed with hybridization solution and incubated overnight with themembrane at 42° C. Following hybridization, the membranes were washedtwice for 5 minutes at room temperature with 2×SSC (NaCl, SodiumCitrate), 0.1% SDS. The membranes were then washed twice for 30 minutesat 42° C. with 0.1 SSC, 0.1% SDS. Membranes were exposed for 1-3 minutesto Biomax ML film (Eastman Kodak, Rochester, N.Y.) for the detection ofchemiluminescent products.

FIGS. 1A and B show the results of the Southern blots. Probescorresponding to DNA sequences that encode BigL repeats hybridized tomultiple DNA fragments in L. kirschneri and interrogans (FIG. 1A). Incontrast, hybridization was not identified with digested genomic DNAfrom the non-pathogenic L. biflexi. Probes based on sequences thatencode for specific C-terminal regions for each of the L. interrogansbigL gene products hybridized to one unique fragment in digested L.interrogans genomic DNA, therefore confirming that there are one copy ofeach of the three bigL gene identified in Example 1 (FIG. 1B). Theseresults illustrate a method of identifying specifically pathogenicLeptospira based on detection of DNA fragments not found innon-pathogenic Leptospira.

Example 2B PCR Detection of bigL Gene Sequences in Leptospira GenomicDNA

This example illustrates the distribution of bigL gene in pathogenicLeptospira. In order to detect bigL genes in other Leptospira species,degenerate primers were designed based on an alignment for bigL genesfrom L. kirschneri strain RM52 and L. interrogans strain Fiocruz L1-130,identified in Example 1. The sequence of the “upstream” primer,designated BigL-1up, is 5′-(GC)AAAGTTG(TC)(AG)(TC)G(TG)CTTGGCC-3′corresponding to positions 46-65 in bigL1 and bigL3 (SEQ ID NO: 1 and5), relative to A of start codon. The sequence of the “downstream”primer, designated BigL-2dn, is5′-(GC)(AT)ACC(AG)TC(CT)GAAAA(AG)AT(AT)CC-3′ corresponding to positions506-487 in bigL1 and bigL3 (SEQ ID NO: 1 and 5), relative to A of thestart codon. Each primer is 20 nucleotides long. These primers weredesigned to anneal to bigL2 at positions 97-116 and 590-571relative tothe A in bigL2's start codon (SEQ ID NO: 3).

PCR reactions were performed with purified genomic DNA from high andlow-pas sage strains of Leptospira. In FIG. 3, amplified DNA fragmentswere identified in PCR reactions with genomic DNA of strains in all fourpathogenic species evaluated. Fragments had the predictedelectrophoretic mobility based on the sequences of bigL1/bigL3 (461 bp)and bigL2 (494 bp). Amplified DNA fragments were not identified in thetwo non-pathogenic Leptospira species evaluated. Therefore this exampleillustrates the application of this PCR method for identifyingspecifically DNA from pathogenic Leptospira in samples.

Example 2C Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)Detection of Leptospira bigL RNA

This example illustrates the detection of bigL RNA in samples. L.kirschneri strain RM52 was grown to late exponential phase, and totalRNA was extracted from 1×10¹⁰ leptospiral cells using the hot-phenolmethod and resuspended in water following ethanol precipitation. ˜2 μgof leptospiral RNA was digested with 6 units of DNase I (Ambion) in 70μl DNase I buffer (10 mM Tris-HCl pH 7.5, 25 mM MgCl₂, 1 mM CaCl₂ in1×RNA secure from Ambion) for 30 min at 37°. To inactivate DNase I, 1.75μl of 25 mM EDTA was added to terminate the reaction, and the enzyme washeat killed for 5 min at 70°. RT-PCR was performed using ˜200 ngleptospiral RNA and Omniscript RT as described (Qiagen). The followingprimers were used to prime the reverse transcriptase reaction:

bigL1, 5′-CGCAGAAATTTTAGAGGAACCTACAG-3′ bigL2,5′-TTTGACTCCAAGACGCAGAGGATGAT-3′ bigL3,5′-ATTTTCAAGATTTGTTCTCCAGATTT-3′; lipL45,5′-ATTACTTCTTGAACATCTGCTTGAT-3′.

The RT reactions were subjected to DNA PCR using Taq polymerase(Qiagen). Prior to PCR, the following primers were added to thereactions:

bigL1, 5′-CTGCTACGCTTGTTGACATAGAAGTA-3′ bigL2,5′-TAGAACCAACACGAAATGGCACAACA-3′ bigL3, 5′-ATCCGAAGTGGCATAACTCTCCTCAT-3′lipL45, 5′-TGAAAAGAACATTACCAGCGTTGTA-3′.

Along with the primers added for reverse transcription, PCR products of500 bp, 479 bp, 440 bp, and 438 bp are expected. To perform PCR, thereaction mixtures were placed in a Techne Progene thermocycler. Aninitial denaturation step of 95° for 1 min was followed by 30 cycles ofdenaturation at 95° for 30 sec, annealing at 53° for 30 sec, andextension at 72° for 30 sec. A final 72° incubation for 30 sec was thenperformed.

The results in FIG. 4 show that RT-PCR method can detect BigL3transcripts and the control LipL46 transcripts. BigL1 and BigL2transcripts were not identified indicating that whereas BigL3 isexpressed in Leptospira, BigL1 and BigL2 may not be. Furthermore, theseresults demonstrate the application of the RT-PCR method to identifyspecific BigL gene transcripts in samples.

Example 3 Expression and Purification of Recombinant BigL Proteins

This example illustrates the use of the DNA sequences of bigL genes toexpress and purify recombinant BigL polypeptides. Two pairs ofoligonucleotides were designed for use in expressing two regions of L.interrogans BigL3. The first region was a region within BigL3corresponding to the 2nd to 6th repetitive domains and corresponded topositions 131-649 of SEQ ID NO: 6 in the L. kirschneri BigL3DNAsequence. Oligonucleotides were designed based upon sequence of lambdaL. interrogans BigL3 clones identified in Example 1 and their sequenceare:

45B-1 5′-ATGGGACTCGAGATTACCGTTACACCAGCCATT-3′ 45B-25′-ATTCCATGGTTATCCTGGAGTGAGTGTATTTGT-3′

PCR amplification with oligonucleotides 45B-1 and 45B-2 and purified L.interrogans genomic DNA was performed to obtain DNA fragments. Thesefragments were digested with XhoI and NcoI Enzymes (New Biolabs) andthen ligated into the pRSETA expression vector (Invitrogen) (16). Thecloned product was sequenced using vector specific primers and primerwalking and the sequence of the 1557 bp product is shown in SEQ ID NO:7. The predicted sequence of the encoded 519 amino acid polypeptide,designated BigL3 region 1, is shown in SEQ ID NO: 8.

A second region was selected for expression that contained the final 200amino acids of the C-terminal region of L. interrogans BigL3. Thisregion corresponded to amino acid positions 1687-1886 of SEQ ID NO: 6 inL. kirschneri BigL3. The oligonucleotides used to clone this region are:

BIGLCTERM1 5′ aac-ctc-gag-cat-aac-tct-cct-cat-aac 3′ BIGLCTERM2 5′ttc-gaa-ttc-tta-ttg-att-ctg-ttg-tct-g 3′

PCR amplification with oligonucleotides BIGLCTERM1 and BIGLCTERM2 andpurified L. interrogans genomic DNA was performed to obtain DNAfragments. These fragments were digested with XhoI and EcoRI enzymes(New Biolabs) and then were ligated into the pRSETA expression vector(Invitrogen) (16). The cloned product was sequenced using vectorspecific primers and primer walking and the nucleotide sequence of the600 bp product is shown in SEQ ID NO: 9. The predicted sequence of theencoded 200 amino acid polypeptide, designated BigL3 region 2, is shownin SEQ ID NO: 10.

Recombinant proteins, rBigL regions 1 and 2, were expressed in BL21(DE3)pLysogen (Invitrogen). Isopropyl-β-D-thiogalactopyranoside (IPTG; 2 mMfinal concentration, Life Technologies) was added to log-phase culturesof E. coli BLR(DE3)pLysS (Novagen) transformed with pRSET plasmidsencoding leptospiral DNA fragments for expression of His6-fusionproteins. 6M guanidine hydrochloride was used to solubilize culturepellets and His6-fusion proteins were purified by affinitychromatography with Ni2+-nitrilotriacetic acid-agarose (Qiagen andPharmacia). The purity of eluted His6 fusion proteins was assessed bygel electrophoresis and staining with Coomassie brilliant blue. Proteinswere dialyzed against PBS, 10% (v/v) glycerol, 0.025% (w/v) sodiumazide. After dialysis, the protein concentration was determined withbicinchoninic acid (42). A Ponceau-S (Sigma Chem Co)-stainednitrocellulose membrane after transfer of purified BigL3 region 1 isshown in FIG. 7. The relative mobility of the purified BigL3 was similarto the estimated molecular mass of approximately 58 kD, which wascalculated based on the predicted amino acid sequence of the recombinantprotein.

Example 4 Example 4A Detection of Antibodies against Recombinant BigLProteins

This example illustrates two among several methods that utilize BigLpolypeptides to detect antibodies in subject samples. Furthermore, thisexample provides methods for serodiagnostic kits for identifyinginfection in subjects suspected of harboring infection.

Immunoblot Detecting of Antibodies to BigL Polypeptides in Samples fromInfected Subjects

Purified recombinant BigL3 region 2 polypeptide (1 mcg/lane) (Example 3)was subjected to sodium dodecylsulfate-polyacrylamide 12% gelelectrophoresis (SDS-PAGE) using a discontinuous buffer system andtransferred to nitrocellulose membranes (Osmomics), as previouslydescribed (17). The nitrocellulose filter was blocked with TBST with 5%skimmed milk, incubated for more than 1 hour with pooled sera frompatients with laboratory confirmed leptospirosis, captured rat (Rattusnorvegicus) reservoirs of Leptospira which had urine and kidney culturespositive for pathogenic Leptospira, and experimental laboratory rats andrabbits, immunized with whole L. interrogans serovar copenhageni strainFiocruz L1-130 lysates. As control experiments, incubations wereperformed with sera from healthy individuals from Brazil, captured ratswho had no culture or serologic evidence for a Leptospira infection andlaboratory rats and rabbits prior to immunization. Sera were diluted1:100 prior to use. After washing, membranes were incubated with goatanti-human gamma chain antibody conjugated to alkaline phosphatase(Sigma), diluted 1:1000, for more than 1 hour. Antigen-antibodycomplexes were detected by color reaction with NBT (0.3 mg/ml) and BCIP(0.15 mg/ml). Pooled sera from leptospirosis patients, captured rats whowere infected with pathogenic leptospires strongly recognized purifiedrecombinant BigL 3 region 1 protein. However, rats immunized with wholeLeptospira lysates did not visibly bind to the BigL3 polypeptide,indicating that although BigL3 is expressed in cultured leptospires(Example 2, FIG. 4), there may be differential expression of the bigL3gene. Sufficient quantities of native BigL3 protein may not be presentin vitro whereas, during natural infection, leptospires in vivo producesufficient quantities of BigL3 to induce a strong immune response.Furthermore, this example illustrates that a spectrum of animals producean immune response to BigL3 during infection and detection of thisimmune response, and detection of antibodies to recombinant BigL3polypeptide can be used as a method to identify infection in subjects.

To further illustrate the use of a detection method for antibodiesagainst recombinant BigL3 polypeptide, an immunoblot evaluation wasperformed with individual sera of patients with laboratory-confirmedleptospirosis, healthy individuals from Brazil and US and patientshospitalized or evaluated in ambulatory clinics with diagnoses otherthan leptospirosis. The microagglutination test and culture isolationwas used to confirm the diagnosis of leptospirosis in patients withclinically-suspected disease (5). The collection of sera fromleptospirosis patients was during five-year surveillance forleptospirosis in the city of Salvador, Brazil. The collection of serafrom control individuals was obtained from pre-existing serum banks ofhospitalized and clinic patients and healthy individuals from Salvador,Brazil and through donations from the Center for Disease Control andPrevention, USA. A list of the sera used is shown in TABLE 1. Seradiluted 1:100 were analyzed following the method described above. Thefinding of any visible colorization of the 1 mcg band of recombinantBigL3 region 1 polypeptide in the immunoblot was considered a positivereaction.

FIG. 8 illustrates that sera from individual leptospirosis patientsreact with recombinant BigL3. Table 1 summarizes the findings thatdemonstrate that more than 90% of hospitalized patients andapproximately 70% of outpatients with leptospirosis react to rBigL3during active infection. All (100%) of the leptospirosis patients reactto rBigL3 during the convalescent-phase of their illness. Table 2compares seroreactivity to rBigL3 with standard diagnostic tests. RBigL3seroreactivity was greater during the initial phase of illness comparedto those observed for standard diagnostic tests. Healthy individualsfrom the US and 88% of the healthy individuals from Brazil do not reactto rBigL3, demonstrating that this reaction to rBigL3 is specific. Thespecificity of the reaction increases to 100% when it is calculatedbased on the frequency of IgM seroreactivity among healthy Brazilianindividuals. Together, these findings illustrate that the method hasutility as a serological marker of active infection and is the basis fora kit that can be used for diagnosis with leptospirosis.

Table 1 also summarizes findings for rBigL3 seroreactivity in endemicregions that have high risk for leptospirosis. 25% of the populationthat resides in these regions demonstrate rBigL3 IgG seropositivity,indicating that this reaction may be a useful marker to identify pastinfection. Among patients with confirmed leptospirosis, 56% wereseroreactive against rBigL3 during the period two years after theirinfection with leptospirosis (Table 2). In the period between 2 and 4years after infection with leptospirosis, 18% demonstrated rBigL3seroreactivity. Together, these findings illustrate that a kit based onthe immunoblot method can detect a past infection with leptospirosis.

Example 4B ELISA-Based Detection of Antibodies to BigL Polypeptides inSamples from Infected Subjects

This example illustrates that ELISA methods are useful in detectingantibodies to BigL polypeptides and in identifying patients withleptospirosis among those with suspected infection. Flat-bottomedpolystyrene microtiter plates (Corning) were coated at 4° C. overnightwith His₆-fusion rBigL3, 0.5-100 ng/well, suspended in 0.05 M sodiumcarbonate, pH 9.6 (16). The plates were washed twice with distilledwater and three times with PBS, 0.05% (v/v) Tween 20 (PBST). Plates wereincubated with blocking solution (PBST/1% [w/v] bovine serum albumin)for 2 hours at room temperature and after four washes with PBST, werestored at −20° C. until use. Wells were incubated with 50 μl of sera,diluted 50 to 200-fold in blocking solution, for 1 hour at roomtemperature with agitation. After four washes with PBST, wells wereincubated with 50 μl of 5,000 to 20,000-fold dilutions of anti-human μor γ-chain goat antibodies conjugated to horseradish peroxidase (Sigma)for 1 hour at room temperature with agitation. Afterwards, plates werewashed twice with PBST and three times with PBS and incubated with 50μl/well of 0.01% (w/v) 3,3′,5,5′-tetramethylbenzidine in substratebuffer (0.03% [v/v] hydrogen peroxide, 25 mM citric acid, 50 mM Na₂HPO₄,pH 5.0) for 20 minutes in the dark at room temperature. The colorreaction was stopped by adding 25 μL 2 NH₂SO₄ and the absorbance at 450nm was measured in an Emax microplate reader (Molecular Devices,Sunnyvale, Calif.).

Initial assays were performed to determine the antigen concentration(mcgs/well) that best discriminated between ELISA reactions of serumsamples from laboratory-confirmed leptospirosis cases (n=4) and healthyindividuals from an endemic area for leptospirosis in Brazil (n=4).Checkerboard titrations were performed with 50, 100 or 200-fold serumdilutions and antigen concentrations per well of 25, 50, 100 and 200 ng.FIG. 6 illustrates that significantly increased absorbance values wereobserved at all serum dilutions and rBigL3 polypeptide concentrationsfor leptospirosis patients than for control individuals.

In subsequent assays to determine sensitivity and specificity, plateswere coated with 50 ng of rBigL3. Incubations were performed with 50 and10,000-fold dilutions of primary sera and secondary antibody conjugate,respectively. Individual serum samples were tested in duplicate and themeans of the two measurements were calculated for analysis. Pairedmeasurements that differed by greater than 10% were retested. Onepositive control serum sample which reacted with all recombinantantigens and one negative control serum sample were included, induplicate, on each plate as a quality control measure. FIG. 7illustrates that leptospirosis patients in the acute phase of illnesshad significantly increased absorbances than control individuals for IgMand IgG seroreactivity (FIG. 7). These differences increased whencomparing absorbance values for patients in their convalescent-phase ofillness. These experiments illustrate that an ELISA-based method fordetecting antibodies against rBigL3 polypeptide is useful foridentifying infection with leptospirosis and can be used as a kit fordiagnosis.

Example 5 Induction of an Immune Response against Leptospira in Subjects

This example illustrates that an immune response against BigL proteinscan be induced via immunization with recombinant BigL proteins. Purifiedrecombinant BigL3 polypeptide derived from L. interrogans was obtainedwith the method described in Example 3. Laboratory rats (Wistar strain)were immunized with 40 mcgs of rBigL3 in Freund's adjuvant (Sigma), andinoculated subcutaneously. Additional immunizations were performed with20 mcgs of rBigL3 at weeks 3 and 6. Blood was collected 7 weeks afterprimary immunization and processed for serum. Immunoblots with rBigL3 (1mcg/lane) were prepared as in Example 4. FIG. 9 illustrates theseroreactivity of rBigL3-immunized rats. rBigL3 was an effectiveimmunogen inducing immunoblot rBigL3 seroreactivity with titers ofgreater than 1:2500 after a total of three immunizations. Furthermore,antibodies raised to rBigL3 polypeptide recognized native antigens inwhole Leptospira lysates (10⁸ leptospires per lane) (FIG. 9). A bandwith relative mobility at 200 kD is faintly stained in immunoblots asare more intensely staining bands with lower relative mobility, whichmay represent degradation of the 200 kD or high molecular weight BigLproteins. Seroreactivity against these native antigens is specific sinceno reactions are observed in the pre-immune sera.

Immunogenicity experiments were performed with purified recombinant BigLpolypeptides derived from L. kirschneri. Purified recombinant proteinswere loaded onto a preparative 12% SDS-PAGE gel and allowed to migrateinto the separating gel by electrophoresis. A band containing 100-200smcg of recombinant protein was excised from the gel, desiccated, groundto powder, dissolved in 1 ml of water, mixed with 1 ml complete Freund'sadjuvant (Sigma), and inoculated subcutaneously and intramuscularly inNew Zealand white rabbits (Harlan Sprague Dawley) that were free ofleptospiral antibodies. Additional immunizations with similar amounts offusion protein in powdered acrylamide gel mixed with incomplete Freund'sadjuvant (Sigma) were administered at four and eight weeks after primaryimmunization. Blood was collected from the rabbits ten weeks afterprimary immunization and processed for serum (Harlow, 1988). Immunoblotswere performed as previously described (Guerreiro et al. Infect Immun2001) with concentrations of 108 leptospires per lane.

FIG. 10. illustrates that immunization with rBigL3 derived from L.kirschneri induces high level antibody titers to native BigL3polypeptides in L. kirschneri and other pathogenic Leptospira speciessuch as L. interrogans. Together these findings illustrate thatimmunization with rBigL polypeptides induces an immune response againstspecies of pathogenic spirochetes other than the species used to designthe recombinant rBigL polypeptide. Furthermore, the antibodies producedby this method of immunization can be used to detect pathogenicspirochetes in samples.

Finally, this example demonstrates that the presence of native BigLpolypeptides is observed in virulent low culture passaged strains andnot in avirulent attenuated high culture passaged strains (FIG. 10).Sera from rBigL3-immunized rabbits recognized a predicted 200 kDacorresponding to BigL3 in whole Leptospira lysates of virulent and notavirulent attenuated strains. This example illustrates that BigLproteins are markers for virulence and that antibodies against BigLproteins can be used as a method to identify virulent strains. SinceBigL may be itself a virulence factor, induction of an immune responseto BigL proteins as demonstrated in the example will be useful forapplication as a vaccine.

TABLE 1 Detection of IgG and IgM antibodies against rBigL and rLipL32 insera from leptospirosis patients and control groups as determined by theWestern Blot method. rBigL3 seroreactivity rLipL32 seroreactivity IgM orIgM or No. IgM IgG IgG IgM IgG IgG Study group tested No. positivereactions (%) Hospitalized cases of confirmed leptospirosis Acute-phase52 37 (71) 46 (88)  48 (92)  22 (42) 21 (50) 38 (73) Convalescent-phase52 19 (37) 52 (100) 52 (100) 21 (40) 45 (86) 46 (88) Outpatient cases ofconfirmed leptospirosis Acute-phase 14  6 (42) 8 (57) 9 (64)  2 (14)  2(14)  3 (21) Convalescent-phase 14  7 (50) 14 (100) 14 (100)  6 (42)  5(36)  8 (57) Healthy individual control groups Non-endemic area (USA) 300 (0) 0 (0)  0 (0)  0 (0) 0 (0) 0 (0) Endemic area (Brazil) 40 0 (0) 5(12) 5 (12) 2 (6) 0 (0) 2 (6) High risk endemic area (Brazil) 40 0 (0)10 (25)  10 (25)   4 (10)  5 (12)  8 (20) Patient control groups Dengue15 0 (0) 0 (0)  0 (0)  0 (0) 0 (0) 0 (0) Lyme disease 15 0 (0) 0 (0)  0(0)  0 (0) 0 (0) 0 (0) VDRL-positive 20 0 (0) 1 (5)  1 (5)  0 (0) 1 (5)1 (5)

TABLE 2 Comparison of the rBigL3 and rLipL32-based Western blot withstandard diagnostic tests for leptospirosis. rLipL32 Standard diagnosticrBigL Western blot Western blot Time evaluation seroreactivityseroreactivity period Median Reciprocal IgM IgM after maximum MAT titerELISA- or or initiation No. reciprocal MAT titer ≧100 IgM IgM IgG IgGIgM IgG IgG of illness tested (range) No. positive reactions (%) Acutephase (N = 52)^(a) 2-6 days 21  200 (0-1600) 12 11 12 16 17  8  8 12(57) (52) (57) (76) (81) (38) (38) (57) 7-15 days 31  400 (0-3200) 17 2025 30 31 14 23 26 (55) (91) (81) (97) (100)  (45) (74) (84) Earlyconvalescent phase (N = 52) 16-21 21  800 (200-12800) 21 15  7 21 21  818 19 days (100)  (100)  (33) (100)  (100)  (38) (86) (90) 21-30 31 1600(0-6400) 31 21 12 31 31 13 27 27 days (100)  (100)  (39) (100)  (100) (42) (87) (87) Late convalescent phase (N = 59) 0-23 25  400 (0-800) 2124  0 14 14  2  2 3 months (84) (96)  (0) (56) (56)  (8)  (8) (12) 24-4717  400 (100-1600) 17  7  0  3  3  2  2  3 months (100)  (41)  (0) (18)(18) (12) (12) (18) 48-78 17  200 (0-800) 15  5  0  3  3  2  1  3 months(88) (29)  (0) (18) (18) (12)  (6) (18) ^(a)Acute-phase serum sampleswere collected upon hospital admission.

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In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. An isolated polynucleotide molecule encoding a polypeptidecomprising the amino acid sequence as set forth in SEQ ID NO: 4 orfunctionally equivalent sequences thereof.
 2. The isolatedpolynucleotide molecule of claim 1, wherein the functionally equivalentsequences comprise an amino acid sequence having at least 80% homologyto the amino acid sequence set forth as SEQ ID NO:
 4. 3. The isolatedpolynucleotide molecule of claim 1, wherein the functionally equivalentsequences comprise an amino acid sequence greater than 90% identical tothe amino acid sequence set forth as SEQ ID NO:
 4. 4. The isolatedpolynucleotide molecule of claim 1, wherein the polypeptide comprisesthe amino acid sequence set forth as SEQ ID NO:
 4. 5. The isolatedpolynucleotide molecule of claim 4, wherein the polypeptide consists ofthe amino acid sequence set forth as SEQ ID NO:
 4. 6. An isolatedpolynucleotide molecule comprising the nucleotide sequence set forth asSEQ ID NO: 3, or functionally equivalent sequences thereof.
 7. Theisolated polynucleotide molecule of claim 6, wherein the functionallyequivalent sequences comprise a nucleic acid sequence having at least80% homology to the nucleic acid sequence set forth as SEQ ID NO:
 3. 8.The isolated polynucleotide molecule of claim 6, wherein thefunctionally equivalent sequences comprise a nucleic acid sequencegreater than 90% identical to the nucleic acid sequence set forth as SEQID NO:
 3. 9. The isolated polynucleotide molecule of claim 6, whereinthe polynucleotide comprises the nucleic acid sequence set forth as SEQID NO:
 3. 10. The isolated polynucleotide molecule of claim 9, whereinthe polynucleotide consists of the nucleic acid sequence set forth asSEQ ID NO:
 3. 11. A vector comprising the isolated polynucleotidemolecule of claim
 6. 12. The vector of claim 11, wherein the isolatedpolynucleotide is linked to a promoter.
 13. A host cell comprising thevector of claim
 11. 14. The isolated polynucleotide molecule of claim 1,wherein the polynucleotide molecule is from a Leptospira species. 15.The isolated polynucleotide molecule of claim 6, wherein thepolynucleotide molecule is from a Leptospira species.